The rapid and accurate detection of trace amounts of organisms such as pathogenic bacteria is important in food and water safety, clinical diagnosis, and military/civilian warfare. Recently, there has been much interest in the identification of various microorganisms due to the increased risks of terrorism via biological warfare agents.
Escherichia coli O157:H7 (E. coli O157:H7) is one of the most dangerous food borne bacterial pathogens. It is commonly found in raw beef, fruits, vegetables, salad bar items, salami, and other food products. Outbreaks of E. coli O157:H7 infections have caused serious illnesses and led to a significant number of deaths. Therefore, in order to prevent accidental outbreaks or intentional terrorist acts, early detection of trace amounts of E. coli O157:H7 as well as other pathogenic microorganisms is critical.
The key requirements for a detection technique to be used for the early detection of microorganisms are specificity, speed, and sensitivity. Conventional detection methods provide qualitative and quantitative information in the presence of substantial amounts of organisms such as bacterial species. However, time constraints and ease of on-site analysis are major limitations because many of these methods rely on the ability of microorganisms to grow into visible colonies over time in special growth media, which may take about 1-5 days. Moreover, detection of trace amounts of bacteria typically requires amplification or enrichment of the target bacteria in the sample. These methods tend to be laborious and time-consuming because of the complicated assay procedures.
Recently, attempts have been made to improve conventional bacterial detection methods to reduce the assay time. One of these efforts has been in the modification and automation of conventional methods. In addition, many developments have evolved to improve detection techniques; for example: direct epiluminescent filter technique (DEFT), mass spectrometry-based methods, and counting and identification test kits. One of the most promising techniques is flow cytometry, which is able to detect 102-103 E. coli O157:H7 cells/mL within 1 hour based on luminescence signal in a flow system. Though the detection time is dramatically reduced, sensitivity improvement is still a challenge.
Development of multiplexed bioassays has recently become an area of rapidly expanding interest and application. Compared to single target detection methods, multiplexed assays reduce the time and cost per analysis, allow for simpler assay protocols, decrease the sample volumes required, and make comparison of samples feasible and measurements reproducible and reliable. Many disease diagnoses and biomedical studies require information from multiple targets such as numerous proteins and genes. Multiplexed assays are thus crucial to complement advances in genomics and proteomics to allow a large number of nucleic acids and proteins to be rapidly screened. Oligonucleotide microarrays and protein arrays can handle a high degree of multiplexed detection using spatially resolved measurements, but the experimental equipment and detection systems are generally not convenient to use on a routine basis, cannot be used to monitor real-time or near-real-time events and cannot be used for biological sample imaging.
Multiplexed microsphere-based flow cytometry assays offer several advantages such as flexibility in target selection, fast binding kinetics and well-controlled binding conditions. Both fluorophores and quantum dots have been embedded into polymer microbeads for high-capacity spectral coding. With the unique advantage of size-tunable emission and broad excitation properties, quantum dots have the potential to be a suitable luminophore for wavelength and intensity multiplexing. However, it is not easy to carry out parallel coding on the nanometer scale.
Because many biological systems, including viruses, membranes, and protein complexes, are natural nanostructures, a need remains for the development of nanometer scale signaling markers with multiplex capability.